During HIV-1 infection, the envelope protein (Env) is presented to the immune system in many forms. In the currently accepted model, the functional form of Env is a trimeric complex of gp120/gp41 heterodimers. Examples of non-functional forms of Env include monomeric gp120, uncleaved gp160 precursor and gp41 from which gp120 has dissociated. The functional trimer has evolved to be a compact structure; indeed, while most monoclonal antibodies (mAbs) recognize only non-functional forms of Env, neutralizing mAbs appear to also bind the functional Env trimer. Env fragments released from particles and infected cells have been considered responsible for the generally poor quality neutralizing response to HIV-1 infection. However, new evidence suggests that infectious HIV-1 particles also bear non-functional forms of Env. In pursuing the goal of developing an HIV-1 vaccine able to elicit potent neutralizing antibodies, we have chosen HIV-1 pseudovirions as model immunogens. In Specific Aim 1, we will use a comprehensive set of techniques in an attempt to generate pseudovirions that exclusively bear functional trimers that are only recognized by neutralizing mAbs. Given the compact, antibody-resistant nature of the trimeric complex, we will expand our studies in Specific Aim 2 to include the receptor-engaged form of Env as an alternative neutralization target. Env-receptor binding induces exposure of otherwise cryptic structures. Although these structures are only transiently exposed in natural infection, they are plausible neutralization targets recognized by well-characterized neutralizing mAbs, exemplified by 2F5. To this end, we have generated a pseudovirion mutant that attaches to susceptible cells but only fuses in a redox-dependent manner. Using this model, we will investigate post-attachment neutralization in HIV-I+ serum. In Specific Aim 3, we will test the immunogenicity of i) pseudovirions bearing only functional trimers and ii) receptor-engaged pseudovirions attached to autologous macaque lymphocytes.